High-throughput Identification of Membrane Protein MAbs (SPECIFIC AIMS FROM PARENT GRANT) Abbreviations: MPA: Membrane Protein Array, MAb: Monoclonal Antibody A. SPECIFIC AIMS (FROM PARENT GRANT) Membrane proteins control the flow of information, nutrients, and signals through the cell membrane, and are the targets for 59% of FDA-approved drugs. Monoclonal antibodies (MAbs) that target membrane proteins can be exceptionally valuable in research, diagnostic, and therapeutic applications, but for the vast majority of membrane proteins there are either no MAbs at all or only poor quality antibodies with limited application. The need for high quality MAbs against membrane proteins has been recognized by industry and the NIH, but efforts to identify such MAbs are limited by the difficulty in expressing and purifying membrane proteins in exogenous systems and by conventional MAb isolation strategies that focus on one target at a time. A novel approach to identify membrane protein MAbs in a high-throughput manner is needed to derive MAbs against the entire human membrane proteome. Integral Molecular is a leader in the field of optimizing membrane proteins and isolating MAbs against them. Here we propose a transformative technology to rapidly isolate and de-orphan large panels of MAbs against membrane proteins in order to create the largest and highest quality catalog of MAbs against membrane proteins currently available. The key technology in this approach is a membrane proteome array (MPA) of structurally-intact membrane proteins expressed in live cells. In Phase 1 studies, we successfully constructed a prototype MPA of 2,639 membrane proteins in ?cell array? format, representing ~50% of the human membrane proteome. We used this MPA to successfully identify the targets for 4 test MAbs of known specificity, including 3 FDA-approved commercial MAbs. We next used the MPA to de-orphan MAbs of unknown specificity, successfully identifying the target membrane protein for 3 out of 6 MAbs screened. We now propose to complete the development of the full MPA representing all ~5,000 human membrane proteins and to use it to create a commercial catalog of MAbs against human membrane proteins. These MAbs will be validated for the most important research applications, and some of them may also have eventual diagnostic and therapeutic application. The MPA platform developed in this project will also be used for a screening service to test the specificity of others' MAbs. Integral Molecular is uniquely qualified for this project because of its experience optimizing membrane protein expression, developing cell array-based technologies, and isolating MAbs against membrane proteins. The Specific Aims of this proposal are: Specific Aim I. Complete and optimize a comprehensive MPA. Here we will complete the MPA to include all ~5,000 human membrane proteins. This will include purchasing additional expression plasmids, optimizing plasmids with low expression, and arraying all plasmids into 384-well microplates for single-step expression in cell arrays. Once prepared, each membrane protein clone will be expressed in live cells and tested for cellular expression by MAb reactivity with an epitope tag on the C-terminus of each protein. Specific Aim II. Identify MAbs against diverse membrane proteins. Here we will use the MPA to generate the largest and highest quality catalog of MAbs against membrane proteins (~1,500 MAbs against ~500 different membrane proteins). We will immunize animals using 5 cell types representing diverse membrane proteins, including neurons, testicular cells, cancer cells, stem cells, and T cells. The planned MAb isolation strategies account for membrane protein conservation (by immunizing chickens, which are genetically divergent from humans), a range of membrane protein expression and immunogenicity levels (using phage selection strategies to isolate diverse and rare MAbs), and long-term manufacturing needs (molecular cloning of each MAb). The specificity of each isolated MAb will be determined with the MPA using a multiplexing protocol for rapid de-orphaning. Each MAb will be selected during isolation to bind its target with high affinity, and will be tested for use in all major research applications, including flow cytometry (the gold standard for high quality membrane protein MAbs), immunofluorescence, immunocytochemistry, and western blot. Year 1 Q1 Q2 Q3 Q4 I. Complete and optimize a comprehensive MPA Complete the membrane protein plasmid library Produce membrane protein arrays Validate and optimize the MPA for expression II. Identify MAbs against diverse membrane proteins Immunize with membrane proteins from diverse cell types Isolate MAbs against diverse membrane proteins De-orphan MAbs on the MPA Validate MAbs for commercial use Year 2 Q1 Q2 Q3 Q4 Year 3 Q1 Q2 Figure 1. Summary of the Specific Aims and expected time-line of this proposal. Green indicates completed Aims.